0.6% 0.6% 0.7% 0.4%1.0% 0.9%

0.4% 0.1% 0.1% 0.4% 0.0% 0.0% 0.0%0.5% 0.2% 0.2%

1.4%

0.1%

2.7%3.6%

3.9%

2.0%

5.3% 5.9%

4.8% 5.6%

3.7%

5.3%

3.1%2.5%

7.9%

3.4%

8.3%

10.5%

6.4%

4.8%

0.0%

2.0%

4.0%

6.0%

8.0%

10.0%

12.0%

Tissue 1 Tissue 2 Tissue 3 Tissue 4 Tissue 5 Tissue 6 Tissue 7 Tissue 8 Tissue 9 Tissue 10 Tissue 11 Tissue 12 Tissue 13 Tissue 14 Tissue 15 Tissue 16 Tissue 17 Tissue 18

ECL QUANTIFICATION BY TRYPAN BLUE STAINING PRE AND POST DMEK PREPARATION

Pre ECL Post ECL

References

Clinical Significance

Introduction

Methods

Results

Quantification of Endothelial Cell Loss in Eye Bank Prepared DMEK Grafts

Using Vital Dye Staining

Authors: Jeff Holiman, CEBT1 , Philip Dye, CEBT1, Christopher Stoeger, CEBT, MBA1

1. Lions VisionGift, Portland, OR, United States

Conclusion

Image 1.

Image 2.

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Disclosures - None

Descemet membrane endothelial keratoplasty(DMEK) is increasing in popularity as a surgicaltechnique to replace dysfunctional cornealendothelium.1 Damage to the cells in the graftarea can easily occur during the delicateseparation of Descemet membrane from theposterior stroma, thereby affecting suitabilityfor transplantation. 2,3 Trypan blue stain 0.06%is currently used in our preparation protocolto visualize the free edge of Descemetmembrane and also is useful to visualize cellswith ruptured cell membranes.4. Due to thefragile nature of this dissection, the objectiveof this study was to determine if anestimation of panendothelial cell loss (ECL) ofthe graft area could be accomplishedconveniently and without compromising graftviability.

Eighteen human donor corneas, 61 y/o agerange 52 to 74 years, with regular endotheliumbut unsuitable for transplant were prepared forDMEK in an eye bank setting under an operatingmicroscope. Trypan blue staining was used tovisualize endothelium that lost viability using acomparison of pre-preparation image to post-preparation image. The central 8mm wasdigitally cropped and ECL was calculated usingthe trainable segmentation feature from theImage J FIJI plug-in package to obtain ahistogram. The histogram was generated byselecting those pixels that appear to bedamaged versus total pixels to estimate ECL.5

Link to short video of LVG DMEK prep:Protocol Summary:

STAIN Cornea-> IMAGE Cornea-> PEEL Cornea-> STAIN Cornea-> IMAGE Cornea-> FIJI Analysis->

ECL Calculation

The combined average net loss over 18 corneasprepared for DMEK in an eye bank setting is 4.6%.It is possible to quantify a percent cell lossincremental to the preparation procedure whichcould be of more value for determining ECD ofDMEK graft immediately before implantation thanstandard techniques of specular with slit-lampmicroscopy alone.

The replacement of dysfunctional endothelium bydonor tissue is the primary goal in endothelialkeratoplasty. This study demonstrates that trypanblue can be used to visualize and quantify areas ofdamaged endothelial cells that may occur duringthe graft preparation in the eye bank. This pan-endothelial analysis can provide anotherperspective about the quality of tissue used forendothelial transplantation. Future studies gearedtoward correlating ECL with clinical outcomes mayhelp us understand the clinical significance ofpreparation related ECL.

Sample of Image Analysis

Data Set

Tissue #4

Tissue #16

Tissue #10

Tissue #13 Tissue #15

Figure 2 A) Greyscale image after trypan blue taken pre-peel B) Fiji Analysis of image pre-peel C) Greyscale image after trypan blue stain taken post-peel D) Fiji analysis of image post-peel

ECL 0.4 %

ECL 2.0 %

ECL 0.4 %

ECL 5.3 %

ECL 0.1 %

ECL 5.6 %

ECL 0.2 %

ECL 10.4 %

ECL 0.2 %

ECL 8.3 %

ECL 0.1%

ECL 7.9% 1.EBAA. 2014 EYE BANKING STATISTICAL REPORT. Eye Bank Association of America;2015.2.Melles GR, Ong TS, Ververs B, van der Wees J. Descemet membrane endothelial keratoplasty (DMEK). Cornea. 2006;25(8):987-990.3.Price MO, Giebel AW, Fairchild KM, Price FW, Jr. Descemet's membrane endothelial keratoplasty: prospective multicenter study of visual and refractive outcomes and endothelial survival. Ophthalmology. 2009;116(12):2361-2368.4.Saad HA, Terry MA, Shamie N, et al. An easy and inexpensive method for quantitative analysis of endothelial damage by using vital dye staining and Adobe Photoshop software. Cornea. 2008;27(7):818-824.5.Jardine GJ, Holiman JD, Stoeger CG, Chamberlain WD. Imaging and quantification of endothelial cell loss in eye bank prepared DMEK grafts using trainable segmentation software. Current eye research. 2014;39(9):894-901.

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Tissue #8

All 18 DMEK grafts were able to be measured. Theaverage cell loss is 4.6% (range 1.6% to 10.4%).Figure 2 demonstrates range of net ECL from1.6% in Tissue #4, 4.9% in Tissue #10, 5.5% inTissue #8, 7.8% in Tissue #13, to 10.4% in Tissue#16.